Process and apparatus for freezing living cells

ABSTRACT

The process for freezing cell suspensions by locating the suspension in a freezing chamber and simultaneously monitoring the temperature of the suspended cells and of the chamber. The cooling of the chamber is regulated at predetermined rates in response to given temperature levels of the sample. The cooling chamber includes a fan, a heater, and a source of refrigerant. The process includes the steps of selectively decreasing and increasing the temperature of the freezing chamber responsive to predetermined temperature points on the freezing curve of the cell sample.

BACKGROUND OF THE INVENTION

The present invention relates to a process and apparatus for freezingliving cells.

In recent years processes for the treatment of cancers and tumors havebeen developed in which, after chemotherapy, the patient is transfusedwith specific body cells. Since it is not possible, because of thedanger of fatal rejection, to use cells of donors other than the patienthimself, it has been necessary to remove the specific body cell from thepatient, prior to treatment and then store such cells until they areneeded. To this end it is necessary to store the withdrawn cells forprolonged periods, often several months. To preserve such cellscryogenic preservation at low temperatures, have been employed since itis yet no other means available to store living cells.

Relatively large quantities of such cells are needed for thepost-therapy infusion and most importantly, such large quantities musthave a relatively large ratio of living cells. Nevertheless, none of theknown cryogenic freezing processes is capable of freezing large cellquantities in a single sample nor are such processes capable ofmaintaining the high levels of living cells required for optimumtherapeutic purposes. While the importance of improved cancer therapymight justify a high cost level, it is almost impossible, no matter atwhat cost, with the current processes to obtain the required largequantity of cells as for example autologous lymphoid blood corpuscles ormedullar cells.

There are several factors in the cryogenic preservation processes whichare extremely important. Among these are the steps of:

(1) The taking of the cell sample from the blood, marrow or tissue andthe concentration therefrom of the required cell type;

(2) The transfer of this concentration to a freezing container;

(3) The mixing of the concentrated sample with a freeze protectant;

(4) The controlled freezing of the specific sample;

(5) Prolonged storage below minus 130° C. (143° K.); and

(6) Thawing of the sample in precise time/temperature relationship; andrevitalization of the cells, i.e., their gradual dilution andelutriation.

Of the foregoing factors the most vital step and the one which has upuntil now presented the greatest difficulty is the controlled freezingof the sample.

According to the known freezing processes the cell samples were placedin closed freezing chambers wherein the temperature was reduced at aconstant rate. It was found, however, that the cooling curve of thesample did not conform to the linear curve of the temperature drop inthe cooling chamber, nor did the cooling curve of the temperature takeinto account the cooling curve of the sample, but to the curve as seenin FIG. 1. The sample would initially follow the curve of thetemperature of the chamber, until a point below the freezing level T_(F)at which time it would rise to a plateau defined at its upper limit bythe freezing temperature T_(F) at its lower limit T_(P) (phasetransformation temperature) constituting a pleateau where it wouldremain for some time. After some minutes, the temperature of the samplewould again drop below the plateau T_(P) at an extremely steep descentuntil it almost reaches the curve of the chamber temperature andthereafter follows in parallel the curve of the chamber temperature.Therefore, freezing processes have been developed which take intoaccount the described abnormal thermal behaviour of aqueous samples.But, these processes don't take into account the actual freezing curveof the freezing samples, i.e. they don't use the sample's temperaturefor the regulatory system. Because of practical problems, the sample'smass is mostly not equal to the mass, the freezing curve has beenestablished for. In this case, the freezing chamber generates a freezingcurve of the samples which may be similar to that shown in FIG. 1,resulting in increased damage to the cells. The slightest variationsfrom the freezing curve of the chamber for the particular celldrastically reduces the number of living cells in the sample andproduces undesirable ice crystals and other harmful effects.Furthermore, all of the freezing units currently available, freezelaboratory samples only, that is, small samples having a volume nogreater than about 2 ml. Consequently, the known freezing systems do notmeet the increased requirements of large quantities of cells for broadspectrum therapy.

To be therapeutically effective, large quantities, as for example in thecase of medullar cells, lymphocyte cells, granulocyte cells, amounts ofabout 1×10¹⁰ (range 1×10⁹ to 1×10¹¹) are required and for thrombocytes(platelets) amounts of about 1×10¹¹ (range 1×10⁹ to 1×10¹¹) arerequired. These increased requirements can not be met by the prior art,since

(a) the cells must be frozen in volumes of about 100 to 200 ml in eachunit sample, as otherwise the loss of time and sterility in fillingsmaller samples is too great. Steritily is insured in the prior artonly, when the techniques of transfusion medicine are used and thestorage the small samples is very expensive. For example, therefrigerant costs for storing a vessel of volume of 320 liters areapproximately 10,400 DM (about $6,000) annually. The present inventionpermits the collection and storage of large amounts and at reducedcosts.

(b) The therapeutic dose must contain after thawing at least 80% livingcells as otherwise a lesser amount of living cells is ineffective incarrying out the vital therapeutic function. In the prior art test cellsare sufficient at 50% viability, while generally, the best of the priorart samples do not produce more than 70% viable cells. On the otherhand, cells frozen by the present invention are viable concentrationsnormally between 80-90% and sometimes to about 95% or more. Of course,not only the viable concentrations of cells give therapeuticeffectiveness, but also the absolute number of living cells, which isthe total recovery. This is the viable concentration times the absolutenumber of cells ready for transfusion divided by the number of cellsoriginally in the sample before freezing. Though a sample may forexample contain more than 70% viable cells, a total recovery may be near10%. When cells are frozen by the present invention, the total recoveryexceeds 80% on an average.

(c) Commercial freezing equipment show temperature fluctuation in thefreezing chamber in about a theoretical value of ±3° C. This fluctuationin temperature reduces the viability of the cells along the edge zonesof the container and reduces the viability even more.

(d) The known commercial freezing units have a temperature/time functionwhich is not influenced by the behavior of the sample itself, but onlyby the behavior of the freezing chamber. Therefore, the freezing in suchequipment will proceed in the desired manner only if the sample volumeis not changed relative to the predetermined setting, and irrespectiveof the actual freezing of the sample itself. Thus, when different samplegeometry is used, a new program control must be employed with suchfreezing units. On the other hand, the control system according to thepresent invention renders such a step unnecessary.

In order to avoid the foregoing difficulties, and to obtain the objectsof the present invention, the present invention proposes to use not onlythe temperature of the freezing chamber itself but a measuredtemperature of the sample being frozen for the control of the variousphases of the cooling process and to determine both the sample andchamber temperatures simultaneously.

SUMMARY OF THE PRESENT INVENTION

According to the present invention, a process of freezing cellsuspensions within a freezing chamber is provided comprising the stepsof lowering the chamber temperature at a first defined cooling rate to agiven temperature value at which time the sample becomes cooled to itsfreezing temperature; holding the temperature of the chamber at thisvalue until the temperature of the sample leaves the phasetransformation plateau and decreases to a second predeterminedtemperature; heating the chamber to an intermediate temperature which isbelow the second predetermined temperature of the sample; maintainingthe temperature of the chamber at the intermediate temperature until atsuch time as 85% of the mass of the sample is present in a frozen state,and; thereafter lowering the chamber temperature at a second definedcooling rate until the temperature at which substantially all the massof the sample is frozen. Only when these relative values are matchedexactly, it is possible to freeze reproducably relatively largequantities of cell suspensions and to obtain after the thaw a highsurvival rate.

Further, according to the present invention there is provided apparatusfor carrying out the foregoing process comprising a freezing chamberhaving room for the mounting of a plurality of containers each having avolume of cells to be frozen, the containers being spaced from oneanother to provide uniform flow of the refrigerant media. The chamberhas an inlet for the refrigerant, a fan for the circulation thereof, anda heater. The chamber defines an enclosed path for circulation of thecooling and heating media. The apparatus further contains a test samplesupplied with a thermo-couple, and a heat sensing probe located in thechamber. In this manner the temperature of the chamber and of thesamples to be frozen is constantly monitored, and the inlet of thefreezing media and the heating media can be continuously andsimultaneously monitored.

This apparatus is provided with a programmed control system wherein theprocess, aforementioned, can be automatically carried out. The programcontrol may be a micro-processor the input of which, relative such aconstant as predetermined cell freezing temperature, etc., can be made,and wherein the operation is made responsive to the actual temperatureof the freezing chamber and samples being frozen.

The present invention further provides novel containers and freezingtrays for the storage and for freezing of relatively large samples.Still further, the present invention provides, through the use of theprocess and the apparatus large concentrations of living cells, having ahigh degree of viability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph, wherein the abscissa t is time in minutes and theordinate T is temperature in degrees centigrade, showing the coolingcurves of the chamber and the samples following the prior art;

FIG. 2 is a perspective view of a sample bag and freezing tray;

FIG. 3 is a schematic drawing of a specific freezing chamber used in theapparatus of the present invention;

FIG. 4 is a schematic drawing of the apparatus of the present invention;and

FIG. 5 is a graph similar to that of FIG. 1 showing the cooling curvesfor the chamber and samples according to the present invention.

DESCRIPTION OF THE INVENTION

Before turning to a description of the present invention it is to bepointed out that a great deal of literature exists on the nature of thecells in question and in particular those characteristics concerningthem during freezing and upon being frozen, namely the rates at whichthe cells cool at a given temperature and time conditions, the plateautemperature of the cells at the freezing state, and the point at whichat least 85% of the cells in any given sample are frozen. It is for thisreason that the present invention does not go into such detail exceptwherein it is necessary for an understanding of the present invention.In speaking of cells in the present disclosure, cells, i.e. corpusclesfound in the liquid portions of the body which can be extracted fromblood, glandular secretions, marrow, as well as tissue cells are to beconsidered.

CELL COLLECTION AND PREPARATION

In general, cells intended for use in therapeutic infusion media areinitially collected by known medical techniques and are separated, asfrom the blood, liquid or tissue, with a known cell separator such as aHaemonetics Mod. 30 or Aminco cell separator under periods of two to sixhours, preferably, however, within an average time of about three tofour hours, or until a sufficient amount of cells is available. forfreezing. Another way of cell separation may be performed by densitygradient centrifugation obtained by centrifugation or gravitationalforce, with or without specific filtering apparatus, without the use ofa cell separator as described above. In many cases, the latter procedureis used to obtain thrombocytes from normal blood units. Theconcentration of the cell component, upon hematocrit should be at leastbetween 20 to 50% and preferably about 35% which may contain ananticoagulant. The hematocrit values are of great importance for theviability and recovery of the cells after the freeze-thaw cycle. This isbecause the cells are dehydrated during freezing and thereby dilute theextracellular medium by the outcoming water. This fact is especiallyimportant for erythrocytes preserved by HES (hydroxyethylstarch), as wasshown in respective investigations. Another important fact is theaggregation tendency of white blood cells and thrombocytes, which isextremely increased when the hematocrit exceeds the desired valuesindicated above. The hematrocrit, the concentration of thecryoprotectant and the cooling rate are dependent on one another, andthis fact is responsible for the drastically decrease in recoveryobtained when the indicated values are not chosen. Tests have shown thatmany living cells, for example, lymphacites, are short-lived in vitroand that damage to these cells already starts before they are subjectedto freezing. The amount of the damage is time dependent. In the priorart, loss of time due to the procedures for transferring the cells,filling predetermined small volume containers, was generallyconsiderable and often lasted more than one hour over the optimun range.As damage to the cells before freezing has a multiple effect on thedamage after thawing, the saving of time in the process of handling thecells leads in itself to an increased cell vitality. As will be seenhereinafter, much of the transfer operation is avoided, according to thepresent invention.

According to the present invention, the cells are collected, i.e. byseparation, in large quantities as for example, bone marrow,lymphacites, granularcites may all be collected in the range of 1×10⁹ to1×10¹¹ cells per single sample, while the thrombocites may be collectedin sample amounts of 1×10¹⁰ to 1×10¹² ; stem cells may be collected inthe range of 1×10⁶ to 1×10⁸ cells per single sample. These corpusclesbeing all cells capable of suspension in a fluid, are then suspended ina volume of approximately 100 to 300 ml of isotonic water or the like,as well as plasma or similar body fluids which may contain ananticoagulant. The volume is actually determined by weighing. Due,however, to the manner in which the corpuscles are obtained, the amountof cell material in any given volume is subject to fluctuation and it isnot known for cell separation what quantity of the required cell isobtained. Therefore, by extracting large volumes of blood marrow or thelike by repeated cell separation, one can be assured that at least asufficient amount of cells is obtained at one time after separation.

Once the required cell quantity is obtained and suspended in its aqueouscarrier, it is further admixed with a freeze protectant and/orstabilizers. Generally, two parts of the cell suspension can be mixedwith 0.5 to 1.5 parts of the freeze protectant, such as a 20 to 40 Tsolution of dimethylsulphoxide (DMSO) in a 60 to 80% amino acid glucosesolution. For erythrocytes cells the freeze protectant may be ahydroxyethyl starch. To this there may also be added an anti-coagulantand stabilizer.

A representative suspension may then be for example 10% DMSO, 23% minoacid glucose solution, 35% cell component, 29% plasma and anti-coagulant3% by weight, based on total weight. The plasma may be body fluid, bloodor aqueous carriers. Another representative suspension may be forexample 10% polyethyleneglycol, 23% aminoacid glucose solution, 35% cellcomponent, 29% plasma and anticoagulant 3% by weight.

Wi th the use of the freeze protectant, the osmotic load of cells isminimized. Further delay in osmotic reaction can be obtained by placingthe cell suspension in a conventional tempering bath.

The collection of such large samples and the suspension thereof inprotective solutions, lends itself to the direct filling of largeflexible bags, such as those seen in FIG. 2 and generally depicted bythe numeral 10, which can be shaped into relatively thin plates having athickness within the range of 3 mm for approximately 100 ml volume to 10mm for 240 ml volume. It has been found that in a thickness of between 4to 10 mm such volumes can be easily and swiftly frozen with almost nodamage to the cells. Secondly, the use of flexible bags enables thesample to be more easily handled before freezing, during the freezingprocess, during thawing, and during the infusion into the patientthereafter; allowing the single bag to be disposed of as a throw-awayitem after use. High sterility is maintained at relatively low cost.Further, such bags may be hermetically sealed by electric welding orheat welding avoiding problems of leakage inherent in the currently usedsheetmetal bottles, sheetmetal plates, sheetmetal containers or othersheetmetal containers which are simply plugged with a stopper. Suitableflexible bags can be formed of polyethylene polyamide, teflon, rapton orpolypropylene components which are generally inert to the plasma bodyfluid and human cell.

THE APPARATUS

For the freezing process itself, the flexible bags are placed in a metalouter tray generally depicted by the numeral 10, as seen in FIG. 2.These trays comprise a bottom and a cover hinged together along one sideand having interleafing side edges. These metal trays are highly heatconductive and have the advantage in allowing manipulation of the bagswithout damage thereto. Furthermore, the metal trays allow the flexiblebag to assume an exact shape optimum for the freezing. That is, theflexible bag may be placed with as large a surface area relative to thedesired thickness indicated above. Thus, the containers themselves mayhave a thickness of anywhere between the 4 to 10 mm range. Forconvenience and greater accuracy in use, a series of containers in theranges set forth above may be supplied with each freezing unit. Thecovered trays are preferably formed of copper sheet of approximately 2mm thickness and may be provided with means for clamping the tray andcover together.

As seen in FIG. 3, the freezing apparatus may be made of an insulatedparallelepiped housing 14, having a hollow interior in which is locateda central insulated or insulating core 16 defining a continuous ovalduct 18; one portion of which defines per se the freezing chamber 20, inwhich are located open shelves or wall flanges on which the collectingtrays 12 may be mounted so as to be spaced one from each other to permitcirculation around and about each tray. An inlet 22 for the introductionof the freezing media such as a source of pressurized liquid nitrogen(LN₂) extends through the housing wall. A fan 24 and suitable baffles 26for equalization of the flow of the media are arranged between the inletand the freezing chamber. A heating unit 28 such as an electric coil,provided with relay switch 29 is located downstream of the freezingchamber. Extending into the freezing chamber is a thermal transducer 30in the form of a probe capable of sensing with great accuracy theinstantaneous temperature of the freezing chamber and converting thesame into an electrical signal.

At least one of the freezing trays 32 is supplied with a pair ofthermocouples 34 extending through the wall thereof so as to contact thecenter of the sample in the flexible sample bag held therein. Thethermocouples are copper-constant thermocouples having a tip diameter of0.5 mm and are fixed by a suitable insulating spacer means through thewall of the reference tray. The reference tray is filled with a flexiblebag containing a suspension consisting precisely of the components ofthe cell suspensions to be frozen except for the absence therein of thecell component itself. In place of the cell component, the referencesample contains an isotonic salt solution. Thus, the reference samplemay contain 10% DMSO, 23% amino acid glucose solution, 35% isotonic saltsolution, 29% plasma, and 3% anti-coagulant or stabilizer. Theadjustment consists of variance detector, PI-section, and pulsegenerator containing the variable pulse-duty factor for theelectromagnetic valve. The reference input is generated digitally andthen converted into an analog signal. The microprocessor used is a modelZ 80 (Zilog, USA) one, in which the user programs are inserted as hardware. The desired parts of the cooling curve are selected by codeswitches in a very easy way thereby avoiding errors of the operator.

The basic control for the refrigeration system according to the presentinvention is shown schematically in FIG. 4. The freezing chamber 18 issupplied through its inlet 22 with a source 38 of liquid nitrogenthrough a hydraulic or electrically controlled valve 36. The liquidnitrogen is maintained under suitable pressure in its container 38(schematically illustrated as a Dewar vessel) by a pressure source 40which may itself comprise nitrogen. The thermocouples 34 from thereference sample 32, and the thermal sensor 30, sensing the temperatrewithin the freezing chamber itself, are coupled to a measuring device42, wherein the temperatures are determined and converted intoelectrical signal outputs. These electrical outputs are fed to acomputer control device 44 comprising a memory storage wherein aselected program (cooling rates, freezing levels, etc.) as required forthe particular sample are entered and stored. The control systemincludes a comparator for converting the electrical signal from thethermocouples and thermosensors with the stored program and an analyzerand adequate means for controlling the operation of the supply valve 36for the liquid nitrogen refrigerant and the operation of the relay 29for the heating unit 28. The control system also includes a recordingdevice by which the actual cooling curve can be visually demonstrated.It will be obvious to those skilled in the art that they or personsskilled in the art of electric or electronic control systems will beable to fashion together the specific details to form a suitablecomputer, recorder, input and control means, etc., without anydifficulty or problem involved. Micro-processor systems, currentlyavailable, may preferably be used.

Before the sequence or cycle of the freezing process begins the desiredcharacteristics of program are entered in the program control by quicklysettng the switches of a simple microprocesser system. This data can, ifdesired, be changed during the freezing process so as to enable thestudy of variations in the cooling curves as they may effect cellvitality. Additionally, fixed programs can be entered which for exampleallow the checking of the existing setting data if necessary.

Strict adherence to the cooling curves of the present process areobtainable throughout the sample and not only at its center but alsonear its edges by employing the platelike containers describedhereinbefore and by providing the continuous oval duct and freezingchamber and flow of the air through the freezing chamber. The flow rateof the refrigerant media can be maintained at about 20 m/s. In order toreduce the lamina boundary layer and/or the turbulent boundary layeralong the surface and edge of the containers, the edges of thecontainers may be provided with special detachable shapes and formsproviding low profiles and air foils. Although the chamber temperaturemay fluctuate about a theoretical value on the average of plus minus0.5° C., this extremely slight fluctuation is not measurable on thesample itself which lies inside the foil itself inside the copper traycontainer. The resolution of the thermocouple devices is ±0.1° C. andsuch fluctuation within the sample itself is not measurable.

The freezing chambers design is such that the refrigerant circulates ina cyclical or oval path. The construction of the freezing chamberpermits the parallel freezing of a plurality of samples which are allpressurized with the flow in the same manner. The homogeneous flow isobtained by additional guide-plates or baffles mounted upstream of thecirculating fan. The supply line for the refrigerant positioneddownstream of the fan is a further reason for the only slightnominal/actual difference in the fluctuation of the chamber temperature.

The actual temperature of the cooling media in the chamber is basicallya function of the LN₂ feed (at predetermined pressure), flow rate (fanspeed) and the concurrent selective use of the heater. By employing anelectric heater, the heater may be turned on and off quickly, and itsheatup and cool-time calculated with a high degree of accuracy. Thus,the selective feed of refrigerant, and use of the heater, sometimesconcurrently can be made to control the chamber and sample temperature,through the sensor probes in each and the computer responsive controlvalves and relays.

Within the limits of the bag volume ranges disclosed and of thecontainer thickness disclosed, the present freezing process is notcritically affected. This is so because the present invention relateschamber freezing rates with the actual temperature of the samples to befrozen by simultaneous sensing of both chamber and sample temperaturesand modifying one relative to the other in response to each.

A second form of container tray may be made from aluminum. Shapedsamples of a volume of 100 ml, aluminum container having a wallthickness of the aluminum of 1 mm (k value of the vessel wall=2950 W/m²° C. The foil bag may have a wall thickness of 0.08 mm. This containermay give the cell suspension to be more frozen a thickness of 3 mm.

THE FREEZING PROCESS

The process of present invention lends itself to the automatic controlby the computer control system previously aforementioned although it isnot limited to this specific form. By pre-storing within the computersystem those known factors such as the freezing temperature, the plateautemperature, the phase transformation temperature as well as 85% frozenmass temperature relating to the desired curve of freezing for the givensample, as well as the initial and secondary cooling rates for thechamber, the system can thereafter be automatically controlled, withoutthe dependence upon time for any particular step to proceed through eachof the successive steps set forth. By the use of control system the datadesired can be changed during the freezing process, to enable the studyof variations in the cooling curves that may effect cell vitality, andto take into account the possible unknown variations in the suspensionsitself. Additionally, fixed programs can be entered to allow thechecking of the existing data during the freezing process per se, ratherthan relying upon the success of automatic control. On the other hand,it will be clear that the use of conventional freezing units may also bemade with more manual controls, to carry out the present invention.

The actual freezing of the samples follows the program set forth in FIG.5 which constitutes the process of the present invention. Samples,generally within the temperature range 0° C. to +32° C., preferably at0° C., inserted in the foil bag and the tray container well dried on theexterior and interior are mounted within the freezing unit as previouslyindicated, and initally the temperature of the freezing unit is loweredat a predetermined rate B_(I) upon which the samples to be frozen followsuit in an almost parallel curve, until the freezing temperature T_(F)is reached. Whereupon, the temperature of the freezing chamber isreduced sharply at an increased rate B_(Ia) until a level T_(U) isobtained. During this short interval the temperature of the sample dipsbelow the freezing point and then sharply rises back to the freezinglevel which is in fact the plateau at which phase transformation takesplace. The phase transformation is defined at the upper level of thefreezing temperature T_(F) and at the lower level by the temperatureT_(P) which is of course slightly lower. The temperature of the chamberis thereafter maintained constant at the temperature level T_(U) untilthe amount of phase transformation in the sample causes the temperatureof the sample itself to dip below the lower plateau limit T_(P). At thistime, the heating unit is activated and the level of the temperature ofthe chamber is caused to sharply rise at a rate B_(Ia) to anintermediate level T_(O) ; i.e., intermediate the lower leveled T_(U)and the temperature of the sample at this point. The temperature of thechamber is again maintained for a period of time at the level T_(O) sothat removal of the latent heat of phase transformation is almostcompleted. During this period the temperature of the sample decreases toa point T_(II) equivalent to the condition wherein the mass of the sameis no longer a radiant source of heat and where at least 85% of the massof the sample is presumed in the frozen state. At this point thetemperature chamber is further cooled at the pre-determined rate B_(II)passing level T_(H), until T_(IIa) when virtually all of the remainingmass is considered frozen. The temperature of the chamber is thereafterreduced sharply along curve B_(IIa) until all of the mass is frozen andreaches its lower most limit, (143K). It is noted that the temperaturelevel T_(O) is less than the temperature level T_(H), preferably by atleast one degree. Upon passing the point T_(H) the heater isdiscontinued. The point T_(H) is of course easily detected by theautomatic control which determines the coolent consumption required tokeep the chamber temperature at the desired temperature. T_(H) is not afixed temperature but depends rather on the actual cooling process;therefore, it is detected automatically.

The freezing point of each of the solutions or suspensions will ofcourse be slightly different. These freezing points are well known andcan be read from standard tables or can be measured osmometrically. (Forexample, for a 10% DMSO solution the freezing point is -4.0° C.). Theautomatic control detects the freezing point of the solution itself bydetecting the plateau temperature of the freezing sample. This actualtemperature is then used to determine T_(P) precisely. This allows theuse of simple thermocouples, which may have a slightly differentoutputvoltage than theoretically desired, which otherwise would resultin misleading temperature measurements. T_(F) is pre-stored in thecontrol system as well, because BI_(a) should start even when the samplesupercools. This method gives accurate cooling curves, thoughsupercooling may appear or not and may not be reproducable. In thisphase i.e., at the freezing temperature, the sample present is of coursein its non-frozen state. The cooling rate B_(I) is also determinablewith respect to the given suspension so that an optimum cooling ratefavorable for the vitality of the particular cells is obtained. It willbe found that, at cooling rate greater or lesser than the optimum,demonstrable damage (thermal shock) occurs, presumably due totransposition within the cellular membrane buildig blocks and theimpairment of the meachanism of the active substance transport (K⁺Na^(+--pump)). The transition temperature B_(Ia) is of course reduced ata rapid rate to minimize the α T_(U) and in order to obtain the desiredlength of the freezing plateau, which is calcuable for a constanttemperature of the chamber (i.e. T_(U)). Rate of -150° C./min or greaterare permissable.

The magnitude of the lower freezing limit T_(U) effects heat transferduring the phase change of the sample, i.e., its freezing state, duringwhich latent heat is released. It should be maintained so that a meanmigration rate of the ice front within the range of 1.00 to 2. mm/min.is obtained preferably the average of 1.5 mm per minute, correspondingto a temperature plateau lasting 1.6 minutes, has proven particularlyadvantageous for 4-10mm thick plates containing white blood cells(lymphocytes, granulocytes, stem cells) or bone marrow cells. As forfreezing of erythrocytes with HES, the migration rate is highlyincreased by lowering T_(u) to 15 to 25 mm/min, preferably to 21 mm/min.The influence of the migration rate has been studied by variation ofthat parameter without affecting the other parts of the freezing curve(that are BI, BII, BIIa). The migration velocities afore mentioned areoptimized values. Deviations from these values resulting in eithersmaller or larger velocities cause damage to the cell to be frozen.Although the action of this effect on the cells is the subject ofresearch, it can be said that the ice front presumably compresses thecells too much at lower values while at higher values the cells may bemechanically damaged by the growth of sharp ice crystals.

The chamber is heated at rate B_(I) b generally at approximately 50° perminute except in the case of freezing erythrocytes with HES, where BI isgiven a value of 400° C./min, by connection the heating unit while theinfusion of the liquid nitrogen refrigerant is either decreased or shutoff completely, until the temperature of the chamber reaches theintermediate temperature T_(O) at which the balance between the heat andthe refrigerant maintains this temperature constantly. Since due to thebinary or ternary nature of the generally aquueous suspensions, thesample continues to form ice by remaining just below the freezingtemperature, the phase transformation plateau is maintained at aconstant temperature during this period, the lower limit thereof T_(P)being easily calculableas T_(P) =T_(F) -0.5° C.

The simultaneous lowering of the temperature of the sample during themaintenance of the chamber temperature at T_(O) results in the furtherconcentration of the residual solution. The level of T_(O) at adetermined difference from that of the temperature of the sample itselfshould be as great as the requirement for latent heat removalnecessitates and for the maintenance of the desired temperature drop inthe sample. It is desirable to shorten the length of these the sample isat the freezing plateau and therefore it is beneficial to optimize heattransfer therein and achieve a corresponding heat transfer during thephase when the chamber is held at T_(O). This is effected by theaccelerated temperature rise of the chamber after passing the phasetransformation temperature T_(P). This optinal in heat transfer resultsfrom the required cooling rate B_(II) and the heat transfer coefficientbetween outer wall of sample and the refrigerant in the interior of thechamber. This coefficient is 110 W/m² ° C., as is seen from measurementson container plates in the freezing chamber. The heat transfercoefficient depends mainly on the evaporation enthalpy of therefrigerant droplets charged in the chamber. Without this evaporationeffect the heat transfer is 80% less, as follows from the calculation ofthe heat transfer coefficient from Nusselt's heat transfer law forsmooth plates at the laminar boundary layer for air. The additionallatent heat is released to the point where the respective eutecticreaction is obtained (a salt water solution -21.2 C.). The eutecticpoint of the respective solution gives the minimum possible temperaturefor the existence of any liquid. Below this temperature, the wholesample is solidified when in thermodynamic equilibrium. Until the samplehas reached an intermediate point between the freezing plateau and theeutectic reaction, i.e., the point T_(IIa), this process plays animportant thermal roll in obtaining the advantages of the presentinvention. Only at this point can the sample be regarded as a frozenbody without any inherent heat source and the cooling only takes plateuntil this point is achieved by stabilizing the chamber at T_(O). Oncethe sample passes through this intermediate point T_(II) the chamber canthen be cooled at a rate, B_(II) until the sample reaches the T_(IIa)level. The heating unit is turned off when the sample reaches T_(H).When the sample temperature is reduced to the point T_(IIa) at whichmost of the cell suspension is frozen, the cooling can be continued atthe more rapid rate BIIa until the conclusion of the cycle. The pointT_(IIa) has been detected in experiments as to be that temperature, atwhich BIIa may be used without additional damage in order to shorten thetime needed for freezing. In all cases, when T_(IIa) is not known, BIIashould have the same value as BII.

From the freezing point T_(F) until the reaching of the point T_(IIa)wherein the cooling sample reaches about -35° C. considerable changes inthe state of the cell and in the original homogenous suspension liquidtakes place, which are of utmost importance for the vitality of thecells. After the solidification temperature T_(F) is reached the newphase ice forms, thereby water is removed from the residual solution andthe concentration of the liquid takes place. The cell reacts withrelease of water, which however does not occur at any desired speed butis limited by the membrane permeability of the cells themselves. Whenfreezing more slowly than specified, the cells remain closed to theconcentrated solution too long, resulting in destruction of the cells asthe shrinkage is too intense and also the proteins are denatured. Whencooling faster than specified, water remains in the cell after the pointT_(II) has been reached. It forms intracellular ice and thereby destroysthe cell from within.

Studies have shown that the effects occur even with minor deviationsfrom the specified cooling rate appropriate for the type of cell. It wassurprising to see that in the process of the present invention one cancool several cell species up to 700° /min. The migration of rate of theice front can be brought (by the action of the length of the temperatureplateau) to the optimum value of the cells other than erythrocytes withHES as a cryoprotectant, to 1,5 mm /min. For erythrocytes with HES, themigration velocity is 21 mm/min. Even those types of cells which arenormally frozen at higher cooling rates can likewise be frozen in acontrolled manner utilizing the present system. Thrombocytes for exampleare frozen at 30° /min (glycerine glucose method), erythrocytes(HES-meflood) are frozen at 700° C./min and action on the temperatureplateau is still possible in this case due to the high maximum coolingrates.

EXAMPLES

(1) Lymphocytes

A sufficient quantity of lymphocyte cells are collected, and suspendedin an aqueous solution of 10% DMSO, 23% amino acid glucose solution 35%cell component, 29% plasma and 3% anti-coagulant stabilizer aspreviously described or in any known manner, and then filled in flexiblebags at about 150 ml volume. These bags are then individually insertedin a copper tray container having an inner thickness of about 5 mm whichare then placed in the freezing chamber. Simultaneously, a referencesample is prepared, identical to the suspension but without the cellcomponent and inserted into the reference copper tray. If the obtainedvolume is for example 80 ml, the same flexible bag may be used asdescribed above. Using a container having an inner thickness of 5 mmdoesn't afford any new settings for the freezing process automaticcontrol, neither does the use of a container of smaller inner thickness,i.e. 3 mm. The use of the smaller thickness gives a better filling ratiofor a single bag. This example is given in order to demonstrate theuniversality of the regulatory system, which therefore has a very broadfield of applications; as concerning to the different volumes described,all examples may be treated in that manner.

(2) Thrombocytes

A sufficient quantity of thrombocytes are collected, and suspended in anaqueous solution of 5% Glycerine, 4% Glukose, 30% plasma, 5% ACD and 56%aminoacid solution including the thrombocytes in a concentration of 800to 1200×10³ cells per microliter. The suspension is filled in flexiblebags at about 100 ml in volume. The bags are individually inserted in acopper tray container having an inner thickness of about 3 mm which arethen placed in the freezing chamber. Simultaneously, a reference sampleis prepared, identical to the suspension but without the cell componentand inserted into the reference copper tray. The freeze protocol is thesame as described under (1), lymphocytes, except for the cooling rate,the freezing point and the plateau end, which may be taken from thetable below.

(3) Granulocytes

A sufficient quantity of granulocytes cells are collected, and suspendedin an aqueous solution of 5% glycerine, 4% glukose, 7% Dextran T10, 23%aminoacid glukose solution, 45% cell component, 23% pLasma and 3%anticoagulant as previously described or in any known manner, and thenfilled in flexible bags at about 150 ml volume. These bags are thenindividually inserted in a copper tray container having an innerthickness of about 5 mm which are then placed in the freezing chamber. Areference sample is prepared simultaneously, identical to the suspensionbut without the cell component. The freeze protocol is the same asdescribed under (1), lymphocytes.

The chamber temperature is initially lowered at rate B_(I) equal to 6°C./min. The sample temperature follows this cooling until the freezingpoint T_(F) of the solution is reached. The freezing point can be readin tables or measured (osmometrically) and is e.g. for the 10% DMSOsolution, etc. of the example -4.0° C. In this phase the sample ispresent in the non-frozen state. Upon reaching the freezing point T_(F),the chamber is rapidly cooled at -150°/min. to T_(U) equal to -55° C.

The end of the phase change at constant temperature is recorded when thepreviously determined temperature T_(P) equal to -4.5° C. is reached.The chamber is now heated at +50°/min by connecting the electric heatingunit until it reaches the temperature T₀ equal to -13° C., andthereafter maintaining this temperature constant until the sample hasreached -12° C. (T_(II)).

Upon the passing of the sample through T_(II), the chamber is cooledfurther at the rate B_(II) equal to 2° C./min until the chamber reachesT_(IIa) equal to about -35° C. At the intermediate point the T_(H) theheating unit is turned off. This occurs automatically by determinationof the required cooling power to remove the heat produced by the heaterafter T_(O) has been reached. At -35° C. the cooling is continued at adecreasing rate equal to 10° C./min to -130° C. This concludes thecontrolled freezing process.

    __________________________________________________________________________              Cooling Cooling Freezing                                                                           Plateau                                                                            Start                                     Cell type rate    rate    point                                                                              end  BII:                                      Example   BI      BII     T.sub.F                                                                            T.sub.P                                                                            T.sub.II                                                                           T.sub.U                                                                            T.sub.O                         __________________________________________________________________________    (2)                                                                             Granulocyte.sup.(a)                                                                   -6° C./min                                                                     -2-3° C./min                                                                   -4° C.                                                                      -4.5° C.                                                                    -12° C.                                                                     -55° C.                                                                     -13° C.                  (3)                                                                             Bone marrow.sup.(a)                                                                   -2° C./min                                                                     -1° C./min                                                                     -4° C.                                                                      -4.5° C.                                                                    -12° C.                                                                     -55° C.                                                                     -13° C.                  (4)                                                                             Thrombocyte.sup.(b)                                                                   -30° C./min                                                                    -30° C./min                                                                    -2° C.                                                                      -2.5° C.                                                                    -20° C.                                                                     -65° C.                                                                     -25° C.                  (5)                                                                             Erythrocyte                                                                           -700° C./min                                                                   -700° C./min                                                                   -2° C.                                                                      -2.5° C.                                                                    -20° C.                                                                     -130° C.                                                                    -90° C.                  (6)                                                                             Medullar.sup.(a)                                                                      -2° C./min                                                                     -1° C./M                                                                       -4° C.                                                                      -4.5° C.                                                                    -12° C.                                                                     -55° C.                                                                     -13° C.                  (7)                                                                             Thrombocyte.sup.(c)                                                                   -6° C./min                                                                     -2-3° C./min                                                                   -1.5° C.                                                                    -2.0° C.                                                                    -10° C.                                                                     -50° C.                                                                     -11° C.                  (8)                                                                             Lymphocyte.sup.(d)                                                                    -4° C./min                                                                     -4-6° C./min                                                                   -2.5° C.                                                                    -3.0° C.                                                                    -13° C.                                                                     -55° C.                                                                     -14° C.                  (9)                                                                             Granulocyte.sup.(e)                                                                   -6° C./min                                                                     -2-3° C.                                                                       -2.5° C.                                                                    -3.0° C.                                                                    -12° C.                                                                     -55° C.                                                                     -13° C.                  __________________________________________________________________________     .sup.(a) DMSO process (10% in the freezing solution)                          .sup.(b) Glycerineglucose process                                             .sup.(c) DMSO process (4% in the freezing suspension)                         .sup.(d) Polyethylene process (10% in the freezing suspension)                .sup.(e) Glycerineglukose-dextran process (5% Glycerine, 4% Glukose, 7%       Dextran T10 in the freezing suspension)                                  

The foregoing table set forth additional examples utilizing the stepspreviously set forth with the given parameters.

The cell suspension frozen according to the examples were stored undercryogenic storage conditions for a lengthy period of time. Upon thawing,at conventional time/temperature parameters and gradual dilution andelutriation, the cell suspension ready for transfusion contains anamount of at least 20% compatible plasma or autologous plasma.Retransfusion of autologous lymphocytes stem cells, and thrombocytesafter freezing by the described process were carried out in 24 cases,after these cancer patients had been treated by chemotherapy. Noproblems due to the cryopreservation of cells arose during or aftertransfusion.

The concentration of viable cells was determined by known in-vitrotests, including fluorescence, hyper blue exclusion specific culturetests, detection of the released cell content and counting or hematocitdeterminations to be as follows:

    ______________________________________                                                                    total recovery                                    Example % of living cells ± stand. dev.                                                                % ± stand dev.                                 ______________________________________                                        (1)     87.6 ± 9.3       83.4 ± 7.3                                     (2)                                                                           (3)                                                                           (4)      88.5 ± 10.6     84.2 ± 8.13                                    (5)     98.7 ± 2.3       75.4 ± 11.2                                    (6)     119.9 ± 44.3     98.3 ± 32.5                                    ______________________________________                                    

While the present invention finds obvious advantage in utilizing theflat tray-like containers, the process may be satisfactorily used forthe freezing of cylindrical samples. The cells for such purposes may asfor test purposes, be suspended in tubes holding a volume of 2 ml ormore. The freezing solution is the same as that described earlier andthere may be however only 1×10⁷ cells per sample. A reference sample inthe form of a tube similarly frozen, filled and provided with a thermalcouple is also employed. Although more extended cylindrical samples canbe similarly cooled by the present process a sample layer thickness inthe main heat releasing direction in excess of 10 mm results in adiminished cell quality value. For this reason therefore the use oflarge cylindrical metal bottles holding 600 ml or more, commonly used inthe preservation or red blood cells, is not suitable for the morecomplicated freezing of other cell components.

It will be seen from the foregoing, that the process by monitoring thetemperature of both the ambient chambers, and the temperature of thesamples permits the use of a flexible control system, by which theadvantages of the present process are obtained, without dependence uponpredetermined time limit. The measured sample temperature is used forcontrolling the various phases of the chamber cooling. Another importantaspect is the use of compensatory heating, with which the heat-upcharacteristic of the chamber is adjusted as needed. This results in thefollowing advantages:

Selection of the duration of the plateau temperature to the optimumvalue given by sample geometry and refrigerant temperature;

Rapid determination of the cooling curve optimal with respect torecovery for a cell type/protectant combination;

Variation of the cooling rate above and below the freezing pointseparately and independently of the duration of the plateau temperature;

Possible changes of single sample mass as well as number of sampleswithout change of program;

Collection of large samples and the elimination of division or fillingup of the cell containers;

Optimum definition of the obtained cells fro the freezing process;

Increased assurance that the samples are sterile.

Various modifications, changes and embodiments have been shown andreferred to in the foregoing disclosure. Others will be obvious to thoseskilled in this art. Accordingly, it is intended that the foregoingdisclosure be taken as illustrated only and not as limiting the scope ofthe present invention.

What is claimed:
 1. Apparatus for freezing cell suspension samplescomprising a freezing chamber having an inlet connected to a source ofrefrigerant media, a freezing section located downstream of said inletfor locating a plurality of separately packaged samples to be frozen, aheating unit located within said chamber downstream of said freezingsection, and a fan located within said chamber between said heating unitand said inlet, a first sensor for determining the temperature of saidfreezing section, and a second sensor for determining the temperature ofat least one of said samples located therein, and control meansresponsive to said first and second sensors for determining the velocityof phase transition of the solid-liquid boundary in the freezing cellsuspension, the actual freezing temperature of the cell suspensionfrozen, and the supercooling of the freezing cell suspension, means forgenerating a freezing curve of the chamber independent of the number ofsamples within said chamber, and means for regulating the flow ofrefrigerant, the heating unit and said fan in a predetermined programresponsive to the instantaneous temperature of the sample, the controlmeans using defined points of the sample temperature to regulate theoperation of said fan, heater and refrigerant inlet to freeze andmaintain said samples in accordance with the freezing curve generated.2. The apparatus according to claim 1, wherein said freezing unitcomprises an oval duct, and said fan and heater are located incommunication with said duct to circulate the refrigerant mediatherethrough, said refrigerant media consisting of solely liquidnitrogen, said duct, fan and heater being arranged so that temperaturedeviations from the mean temperature in the chamber are smaller that 2°C. and preferably 0.5° C. during cooling, whereby, temperaturedeviations inside the samples due to this effect larger than 0.1° C. areavoided and so that a temperature of -190° C. or higher is maintainedduring the freezing process and the thermal heat transfer coefficient isincreased during cooling by vaporization of the droplets of liquidnitrogen on the outer surface of the containers contaning the samples,said droplets being generated by the fan whereby the samples aremaintained having a desired homogeneous temperature field internally. 3.The apparatus according to claim 1, wherein said control means iscapable of determining temperature differences of 1/10° C. or smaller,of any kind of supercooling of samples down to -100° C., and of anyfreezing temperature of the samples in the range of 0 to -100° C. 4.Apparatus for freezing cell suspension samples comprising:a housinghaving walls defining therein an enclosed oval duct forming acontinuously circulating passage, one section of said duct constitutinga freezing section for the location of a plurality of separatelypackaged samples to be frozen and having, an inlet to said passagespaced from said freezing section, for the introduction of a refrigerantinto said duct, a heating unit located in said duct between said inletand said freezing section downstream of said freezing section forheating said refrigerant, a fan located in said duct between said inletand said freezing upstream of said freezing section for moving saidrefrigerant through said freezing section, a first sensor fordetermining the temperature within said freezing section, a secondsensor for determining the temperature within a selected one of saidsamples, and control means for regulating the flow of refrigerant, theoperation of said heating unit and said fan responsive to said sensorsin a predetermined program to effect the desired freezing of saidsamples.
 5. The apparatus according to claim 4 including baffle meanslocated in said duct for uniformly distributing said refrigerant in saidfreezing section.
 6. The apparatus according to claim 4 including meanslocated in said freezing section for holding a plurality of samples tobe frozen spaced from each other to permit movement of mediatherebetween.
 7. The apparatus according to claim 4 wherein each of saidsamples is sealed in a hermetically sealed impervious foil bag containedin a flat rigid parallelepiped container having an overall innerthickness of between 4-10 mm.
 8. The apparatus according to claim 7wherein said second sensor extends within said sample bag.
 9. Apparatusfor freezing cell suspension samples comprising a housing having definedtherein a freezing section, an inlet for the introduction of refrigerantinto said freezing section, a heating unit for heating said refrigerant,a fan for moving said refrigerant through said freezing section, a firstsensor for determining the temperature within said freezing section, asecond sensor comprising a thermocouple detecting the velocity of thephase transition in the liquid-solid boundary of the selected sample,the actual temperature of the sample and any supercooling of the sampleand control means for regulating the flow of refrigerant, the operationof said heating unit and said fan responsive to said sensors in apredetermined program to effect the desired freezing of said samples.10. The apparatus according to claim 5 wherein said control meansincludes programable means for generating a freezing curve for operationof said unit, heater and fan whereby said samples have optimal phasetransition, independent of the number of separate samples in saidfreezing chamber.